Detection of bovine viral diarrhea virus by TaqMan® reverse transcription polymerase chain reaction
Detection of bovine viral diarrhea virus by TaqMan® reverse transcription polymerase chain reaction
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DOI：
10.1177/104063870201400205
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发表时间：
2002-03-01
影响因子：
1.5
通讯作者：
Faaberg, KS
中科院分区：
文献类型：
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作者：
Mahlum, CE;Haugerud, S;Faaberg, KS
牛病毒性腹泻病毒（BVDV）持续感染的小牛和奶牛的检测和消除对该病原体的控制具有重要意义。从历史上看，BVDV检测涉及细胞培养分离，然后通过免疫荧光或免疫过氧化物酶单层测定（IPMA）方法检测病毒。最近，免疫组织化学（THC）已被添加为BVDV检测的常规测试。基于凝胶的逆转录聚合酶链反应（RT-PCR）检测BVDV比细胞培养分离更敏感和快速，但检测结果可能受到核酸扩增过程中样品污染的影响。本研究旨在建立一种封闭管格式的BVDV核酸扩增和检测方法TaqMan(R) RT-PCR。将新技术的结果与病毒分离、IPMA和免疫组化的结果进行了比较。TaqMan(R) RT-PCR检测结果显示，除IHC阳性1例外，IPMA阴性、IHC阴性和病毒分离阴性的样品均检测到BVDV。TaqMan(R)闭管式RT-PCR为BVDV检测提供了一种快速、经济、高容量和敏感的方法，而无需担心与开管凝胶PCR检测相关的扩增cDNA产物污染。
Detection and elimination of calves and cows persistently infected with bovine viral diarrhea virus (BVDV) is important for the control of this pathogen. Historically, BVDV detection involved cell culture isolation followed by virus detection through immunofluorescence or immunoperoxidase monolayer assay (IPMA) methods. More recently, immunohistochemistry (THC) has been added as a routine test for BVDV detection. The detection of BVDV by gel-based reverse transcription polymerase chain reaction (RT-PCR) is more sensitive and rapid than by cell culture isolation, but test results can be compromised by sample contamination during nucleic acid amplification. This study was designed to develop a closed-tube format of BVDV nucleic acid amplification and detection, TaqMan(R) RT-PCR. The results of this new technique were compared with those obtained with virus isolation, IPMA, and IHC. With TaqMan(R) RT-PCR, BVDV was detected in many samples negative by IPMA, IHC, and virus isolation with the exception of I sample that was positive by IHC. TaqMan(R) RT-PCR in a closed-tube format offers a rapid, economical, high volume, and sensitive method for BVDV detection without the concerns of amplified cDNA product contamination associated with open-tube gel-based PCR tests.